An HPLC typically contains two columns: an analytical column, which happens to be answerable for the separation, in addition to a guard column that is definitely put ahead of the analytical column to protect it from contamination.
각각 다른 산업 분야에 대한 자세한 정보 및 다양한 카테고리는 다음 써모 피셔 사이언티픽 학습 센터에서 산업 및 응용 과학 페이지를 확인하세요.
The sample separation happens while in the column for which temperature ought to be continual. So to keep up the consistent temperature, a column is positioned inside the column oven. The conversation of the person elements plus the stationary stage begin to come about. Should the stationary period as well as people today contain the exact same nature, i.e., both of those are polar, then the polar compound will communicate with it for some time.
. Once we look at the chromatograms from these 7 mobile phases we may possibly see that one or more offers an satisfactory separation, or we may discover a region within the solvent triangle where by a separation is possible.
Separation Mechanism: Unique column chemistries offer unique separation mechanisms based on analyte Qualities like dimensions, polarity, or charge. Being familiar with the analytes and sought after separation mechanism guides column collection.
A detector identifies and steps Every component. Retention time indicates time taken for every compound to exit the column. HPLC's efficiency depends on factors like check here column form and cellular phase composition. Common routine maintenance makes sure precise effects. Knowing HPLC's step-by-phase approach is significant for specific chemical Examination in laboratories.
In liquid–liquid chromatography the stationary period is really a liquid film coated with a packing substance, commonly 3–ten μm porous silica particles. Because the stationary phase can be partially soluble within the mobile period, it may well elute, or bleed in the column over time.
前述した従来の順相タイプに対して、逆相クロマトグラフィーにおいては固定相に低極性のもの（例えばシリカゲルにアルキル基を共有結合させたもの）を、移動相に高極性のもの（例えば水や塩類の水溶液、アルコール、アセトニトリルなどの有機溶媒）を用いる。また珍しいケースではあるが、分離のための移動相pHをシリカゲルの使用範囲から外れたところに設定する必要がある場合、あるいはシリカゲル表面に残っている未反応シラノール基が分離に悪影響を及ぼし、かつそれが移動相の変更によっても解決できない場合には、固定相として樹脂を用いることがある。分析物はより極性の低いほどより強く固定相と相互作用して溶出が遅くなる。また極性の低い物質の割合が多い移動相ほど溶出が早くなる。
Lousy resolution usually means analytes elute much too near together, creating them difficult to tell apart. This is the best way to troubleshoot:
Maximize or lower the ionization point out of analytes, affecting their affinity for that stationary phase.
Modifying the mobile section’s get more info polarity index variations a solute’s retention variable. As we figured out in Chapter 12.3, nevertheless, a change in k is not really a successful way to boost resolution when the Original worth of k is greater than ten.
In reversed-phase HPLC the buy of elution is the other that in a traditional-section separation, with much more polar solutes eluting initially. Expanding the polarity on the mobile period causes more time retention moments. Shorter retention occasions demand a cellular period of lower polarity.
Mobile section impurities: Contaminants in the cell period can elute through the column and demonstrate up as ghost peaks. Prepare a fresh cell stage with high-purity solvents and contemplate filtering the cell period ahead of use.
An inside common is necessary when applying HPLC–MS since the interface involving the HPLC plus the mass spectrometer won't let for any reproducible transfer of the column’s eluent in the MS’s ionization chamber.